Transcription factors represent a group of molecules within the cell that function to connect extracellular signals to intracellular responses. Immediately after an environmental stimulus, these proteins which reside predominantly in the cytosol are translocated to the nucleus where they bind to the promoter regions of various genes and activate the transcription of the respective target gene. Certain transcription factors belong to a group of genes which are transcribed without any de novo protein synthesis allowing quick response times to external stumuli. These transcription factors are usually expressed at low basal levels but they can be dramatically induced in a variety of cell types by many different stimuli, in which the quantity of transcription factor and the DNA binding activity also rise.
One such class of transcription factors is the activator protein-1 (AP1) transcription factor family. These factors are induced by a variety of signals, including those eliciting proliferation, differentiation and programmed cell death (apoptosis). AP1 is a dimeric complex consisting of either a homo or heterodimer of Jun family proteins (c-Jun, JunB or JunD) or a heterodimer of one Jun member and one member of the Fos family of proteins (c-Fos, FosB, fra-1 or fra-2). Modulation in Fos and Jun expression levels will define the AP1 dimer formed and hence the potential influence the transcription factor has on target genes. Numerous cellular and viral genes contain AP1 binding sites within their promoters and, accordingly, AP1 has been shown to play a role in the regulation of both basal and inducible transcription of these genes.
Fra-1 (also known as FOSL1, fos-related antigen 1 and fos-like antigen 1) encodes a serum-inducible protein that is antigenically related to Fos and is the protein product of a gene in the group of immediate early genes which bind the Jun family members to form the AP1 transcription factor. Fra-1 exhibits transcriptional induction within 60 minutes of mitogen stimulation and has been shown to contribute to the DNA-binding activity ascribed to AP1 (Matsui et al., Oncogene, 1990, 5, 249-255). It has been shown that fra-1 can both activate and repress transcription of target genes through separate functional domains in the protein (Gius et al., Mol. Cell. Biol., 1990, 10, 4243-4255). Specific examples include the induction and repression of atrial natriuretic peptide in the myocardium (Kovacic-Milivojevic and Gardner, Hypertension, 1995, 25, 679-682) and induction of the involucrin promoter in epidermal cells (Welter et al., J. Biol. Chem., 1995, 270, 12614-12622; Welter and Eckert, Oncogene, 1995, 11, 2681-2687). In rat PC12 cells, it was shown that constitutive expression of fra-1 inhibits growth factor-induced cell differentiation (Ito et al., Oncogene, 1990, 5, 1755-1760).
The role of fra-1 in the development of cancer has been the most broadly investigated. The fra-1 gene has been localized to chromosome 11q13, a genomic region previously implicated in several neoplastic disorders including breast, esophageal, hepatocellular, bladder and hematopoietic cancers (Sinke et al., Genomics, 1993, 18, 165).
Several studies have implicated deregulated fra-1 expression in the development of neoplasia. In the rat, fra-1 overexpression in fibroblasts results in anchorage-independent growth in vitro and in tumor development in athymic mice (Bergers et al., Mol. Cell. Biol., 1995, 15, 3748-3758). Furthermore, in rat thyroid cells, the inhibition of fra-1 by stable transfection of a fra-1 antisense vector significantly reduced the malignant phenotype of transformed cells (Vallone et al., Embo. J., 1997, 16, 5310-5321). In the Eker rat model of renal carcinogenesis (RC), fra-1 is overexpressed as are other AP1 proteins. In these studies, it was shown that transfection of antisense oligonucleotides targeting fra-1 and other AP1 proteins reduced the growth of RC cells. The single antisense sequence chosen for each was a 15 base pair phosphorothioate sequence targeting the AUG translation initiation codon of rat fra-1 (Urakami et al., Biochem. Biophys. Res. Commun., 1997, 241, 24-30). Studies of mouse adenocarcinoma cells, indicated that overexpression of fra-1 induced transformation and increased invasiveness, suggesting a critical role for fra-1 in more than one step of tumor progression (Kustikova et al., Mol. Cell. Biol., 1998, 18, 7095-7105). Finally, in human breast cancer cell lines differing in their estrogen receptor (ER) status, it has been shown that fra-1 expression levels modulate AP1 activity induced by estradiol. These studies showed that overexpression of fra-1 in ER-containing cells decreased the positive effects of estradiol and inhibition of fra-1 in ER-deficient cells using a fra-1 antisense expression vector abolished the negative effects of estradiol. These studies suggest that the pharmacological modulation of fra-1 activity or expression may be an appropriate point of therapeutic intervention in pathological conditions such as cancer (Philips et al., Mol. Endocrinol., 1998, 12, 973-985).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of fra-1. To date, investigative strategies aimed at modulating human fra-1 function have involved the use of antisense expression vectors.
Other potential strategies involve the administration of compounds that induce fra-1 expression. It has been suggested that certain polyphenols like those found in green teas, may have therapeutic value by activating fra-1 expression thereby repressing AP1 transactivation. These compounds have been shown to reduce tumor growth in some animal studies (McCarty, Med. Hypotheses, 1998, 50, 511-514). It has also been demonstrated that fra-1 is a target for retinoid regulation. Studies by Kaiser et al. have shown that retinoid-mediated induction of fra-1 might function as a negative regulator of AP1 activity in human pancreatic carcinoma (Kaiser et al., FEBS Lett., 1999, 448, 45-48).
However, these strategies are untested as therapeutic protocols and consequently, there remains a long felt need for additional agents capable of effectively inhibiting fra-1 function.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of fra-1 expression.
The present invention provides compositions and methods for modulating fra-1 expression.